Genotoxic effects of 8-hydroxylquinoline in loach (Misgurnus anguillicaudatus) assessed by the micronucleus test,comet assay and RAPD analysis

This study was a preliminary step in evaluating the genotoxic effects of 8-hydroxylquinoline (8-HOQ) in loach (Misgurnus anguillicaudatus) using the micronucleus, comet and RAPD assays. In the micronucleus test and comet assay, the micronuclei rate (%) and three comet parameters (trailing rate, tail length and tail moment) in treated fish increased with increasing 8-HOQ concentration and treatment time. These results showed that exposure to 8-HOQ significantly induced genetic toxicity in loach blood cells. A subsequent RAPD assay also showed that 8-HOQ induced a genotoxic effect in loach. Among the 23 tested RAPD primers, 11 primers produced unique polymorphic band patterns and generated RAPD profile variations that displayed the band intensity, disappearance of bands and appearance of new bands of amplified DNA in the 8-HOQ-treated genomic DNA samples. In addition, the variation in RAPD profiles was time- and concentration-dependent. These results suggested that 8-HOQ is potentially harmful to fish and may be a toxic contaminant in the aquatic environment.     

Use of RAPD to detect sodium arsenite-induced DNA damage in human lymphoblastoid cells

Inorganic arsenic is a known human carcinogen, yet its mechanism of action remains unclear. Our previous study showed that arsenite significantly induces oxidative DNA adducts and DNA-protein cross-links in several mammalian cell lines. In the present study, we used the random amplified polymorphic DNA (RAPD) assay to evaluate the possible target in the genomic DNA of human lymphoblastoid cells that were exposed to sodium arsenite. Treatment with both 10 and 80 microM arsenite for 4h induced significant changes in RAPD profiles compared with the control pattern. Two 10-mer RAPD primers (D11 and F1) produced the most distinguishable banding profiles between arsenite-treated and control genomic DNA. The sequencing of four arsenite-sensitive RAPD bands showed that the RB1CC1 and PACE4 genes might be the DNA targets of sodium arsenite treatment. We propose that arsenite may induce sequence- or gene-specific damage and then change the RAPD profile in human lymphoblastoid cells. The results of our study also show that RAPD combined with other techniques is a good tool for detecting alterations in genomic DNA and for the direct screening of new molecular markers related to arsenite-induced carcinogenesis.     

Application of random amplified polymorphic DNA (RAPD) to detect genotoxic effect of trifluralin on maize (Zea mays)

Trifluralin is a widely used dinitroaniline herbicide throughout the world. However, limited efforts have been made to study its genotoxic effects on different plants. The present study aimed to evaluate the herbicide’s genotoxic potential on maize (Zea mays) by using the random amplified polymorphic DNA (RAPD) technique. For this purpose, maize seedlings were treated with aqueous solutions of trifluralin at concentrations ranging from 0.5 to 3 ppm for 7 days. In the RAPD analyses, 15 primers were used and 91 bands were obtained, with an average of 6.06 bands per primer in the control seedlings. After trifluralin treatment, significant changes were observed in RAPD profiles. These changes included loss of normal bands and appearance of new bands, in comparison to the control group, and they were dose dependent. In addition, root growth and total soluble protein level in trifluralin-treated seedlings were analyzed and compared for genomic template stability (GTS), which was performed for the qualitative measurement of changes in randomly amplified polymorphic DNA (RAPD) profiles. The results showed that GTS, root growth, and total soluble protein content of the seedlings gradually decreased with an increase in trifluralin concentration. These findings suggest that the RAPD technique is a useful biomarker assay to evaluate the genotoxic effects of herbicides on plants.     

Investigation of quinocetone-induced genotoxicity in HepG2 cells using the comet assay,cytokinesis-block micronucleus test and RAPD analysis

Quinocetone, a new quinoxaline 1,4-dioxide derivative, has been approved as an animal growth promoter in China since 2003. To investigate the genotoxicity of quinocetone in vitro, its effects on the extent of DNA injury in human hepatoma (HepG2) cells accompanied by chromosomal damage and genomic DNA alterations were tested. The cell viability test indicated that quinocetone inhibited cell proliferation as a function of dose and time. In the comet assay, significant DNA fragment migration was observed in a dose-dependent manner. A dose-dependent increase of the micronucleated (MN) cell frequency was shown in cytokinesis-block micronucleus (CBMN) test. The gain/loss of randomly amplified polymorphic DNA (RAPD) bands and the change of band intensity in RAPD profiles were obtained after HepG2 cells were exposed to quinocetone at concentrations of 1.25, 2.5 and 5 μg/mL. The results demonstrated that quinocetone exerted genotoxic effects on HepG2 cells. Thus, the use of quinocetone as a growth promoter in animal feed should be seriously considered.     

A random amplified polymorphic DNA (RAPD) primer to assist the identification of a selected strain, aizu K-111 of Panax ginseng and the sequence amplified

Panax ginseng is widely used as a Chinese medicine, but it takes a long time to reach harvest and to establish its qualified strains. In the course of searching high quality Panax ginseng, we found a useful random amplified polymorphic DNA (RAPD) primer, which showed a 725 base pair band for a selected elite strain Aizu K-111 (now called Kaishusan) including its cultured tissues, while the other strains did not necessarily show this band. We sequenced the DNA fragment amplified and designed primers to improve electrophoretic profiles, based on the sequence.     

不同产地白及遗传多态性的RAPD分析

目的 研究国内不同产地白及的遗传多样性和亲缘关系。方法 利用随机扩增多态DNA （random amplified polymorphic DNA,RAPD）引物,RAPD分子标记技术分析国内50个不同产地的白及。结果 RAPD技术扩增产物经琼脂凝胶电泳检测,从50条引物中筛选出10条（S8,S9,S14,S19,S23,S25,S28,S29,S30,S31）条带清晰的引物,10条引物共扩增出88条DNA条带,其中多态性的DNA条带数目为81条,占总数的92.05%。每个引物能扩增出5~11条DNA条带,平均可扩增出8.8条;扩增最少的引物为S19,扩增出5条DNA条带;扩增最多的引物为S29,扩增出11条DNA条带。而每个引物能扩增的多态性DNA条带数为3~11条,平均可扩增出8.6条。结论 不同产地的白及具有较丰富的遗传多样性,RAPD可有效应用于白及的遗传多样性研究。     

铁皮石斛野生居群遗传多样性的RAPD分析与鉴别

目的采用RAPD分子标记技术，对铁皮石斛8个野生居群的遗传多样性、亲缘关系以及分子鉴别等进行研究。方法筛选随机引物进行RAPD分析，通过UPGMA聚类，研究铁皮石斛各居群间的遗传关系，构建居群亲缘关系的分子系统树；利用特异性条带对铁皮石斛野生居群进行指纹分析鉴别。结果共筛选出10个有效引物，在8个野生居群材料的RAPD扩增中共得到439个位点，平均每个引物扩增出43.9个位点，每个居群扩增出54.9个位点；在所获得的104条可重现谱带中，9条是单态的，95条是多态的，多态性程度达91.35%，遗传距离在0.590～0.727之间，平均为0.686。结论铁皮石斛居群间遗传差异明显，具有较丰富的遗传多样性；RAPD可以作为铁皮石斛野生居群遗传多态性、居群亲缘关系和分子鉴别研究的有效手段；引物S412可以有效鉴别铁皮石斛的8个野生居群。     

Choice of Methodology for Assessing Genetic Impacts of Environmental Stressors: Polymorphism and Reproducibility of RAPD and AFLP Fingerprints

PCR-based multi-locus DNA fingerprints represent one of the most informative and cost-effective measures of genetic diversity and are useful population-level biomarkers of toxicologic and other anthropogenic impacts. However, concerns about reproducibility of DNA fingerprints have limited their wider use in environmental biology. We assessed polymorphism and reproducibility of two common fingerprinting techniques, RAPD (randomly amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism), in pedigreed populations of rainbow trout (Oncorhynchus mykiss) to derive general rules for selective removal of problematic fingerprint bands. We found that by excluding bands that comprised less than 1% of total intensity, and by excluding the largest and smallest 10% of the bands, we could achieve nearly 100% reproducibility of AFLP fingerprints. Similar application of band exclusion criteria to RAPD fingerprints did not significantly enhance their reproducibility, and at least 15% of RAPD bands were not fully repeatable, heritable, or transmittable. The RAPD technique produced more polymorphic fingerprints than AFLP; however, considering that a substantial proportion of RAPD markers did not demonstrate Mendelian inheritance patterns, the AFLP methodology is to be preferred for future research.     

Comparison of ultraviolet-induced genotoxicity detected by random amplified polymorphic DNA with chlorophyll fluorescence and growth in a marine macroalgae, Palmaria palmata

The random amplified polymorphic DNA (RAPD) technique was used to detect DNA damage in the sublittoral macroalgae Palmaria palmata (Rhodophyta) exposed to both ambient and elevated irradiances of UV-B (280-315 nm). To investigate the potential of this method in ecotoxicological assessments, the qualitative and quantitative modifications in RAPD profiles were compared with changes in a number of physiological and fitness parameters. RAPD detectable modifications in DNA profiles were observed in all UV exposed individuals compared with controls. Changes in chlorophyll fluorescence (F(v)/F(m) ratio), in vivo pigment absorptance, thallus growth and RAPD profiles, examined simultaneously, provided a sensitive measure of UV-induced toxicity. In conclusion, the application of the RAPD method in conjunction with other suitable physiological and fitness measurements, may prove to be a valuable tool for investigating the specific effects of genotoxic agents upon marine algal populations. Ultimately, this methodology may allow the ecotoxicological examination of the link between molecular alterations and measurable adverse effects at higher levels of biological organisation.     

RAPD方法在细辛类药材鉴别研究中的问题及其对策

以细辛类药材的鉴别为例,对RAPD方法在药材鉴别中所存在的问题进行了探讨,结果表明药材DNA模板浓度,降解程度及药材的产地均对RAPD产物有不同程度的影响,从而提出选择适宜的药材DNA模板浓度,筛选合适的引物和采用对照组聚类分析等方法来消除上述因素的影响,进而讨论了RAPD方法在药材鉴别上的适用范围。     

Evaluating wastewater-induced plant genotoxicity using randomly amplified polymorphic DNA

Wastewater often contains genotoxic substances that can resist different stages of the treatment process. In the present study, randomly amplified polymorphic DNA technology was applied to evaluate the genotoxic effects of wastewater (treated and raw) irrigation on oat plants (Avena sativa). RAPD profiles obtained showed that both treated and raw wastewater (RWW) were having genotoxic effects on oat plants. This was apparent by the appearance/disappearance of bands in the treatments compared with the control plants. From the 15 primers used, 186 bands were obtained with an average of 12.4 bands per primer. Irrigating plants with RWW caused 51 new bands to appear and 19 to disappear. Treated wastewater (TWW) caused only 16 new bands and the loss of 17 bands. This makes TWW less genotoxic than RWW. The Euclidean distances shown on the dendrogram, revealed the presence of two clusters according to dissimilarity values. One cluster contained the control plants and those irrigated with TWW, whereas the second contained the plants irrigated with RWW. Similarity indices calculated between the treatments and the control plants showed that the control and the plants irrigated with TWW had a similarity index of 0.87, the control and plants irrigated with RWW 0.73 and between the treatments 0.75.     

Utilization of RAPD markers to assess genetic diversity of wild populations of North American ginseng (Panax quinquefolium)

The Catskill Mountains of New York State are an important source of wild-collected American ginseng (Panax quinquefolium) and, increasingly, of woods-cultivated ginseng. The objective of this study was to assess genetic diversity among 9 different wild ginseng populations in and adjacent to the Catskill Mountain region of New York State and to compare these to wild populations from other states including Kentucky, Tennessee, North Carolina, Pennsylvania, and Virginia, and one cultivated population from Wisconsin. Randomly amplified polymorphic DNA (RAPD) markers were used to estimate the genetic distance among samples from the 15 populations. Pooled DNA from 10 plants of each of 8 New York populations was initially screened with 64 random primers; subsequently, the 15 primers that exhibited the greatest number of reproducible polymorphic markers were selected for further experimentation. Gel electrophoresis with the selected 15 primers produced 124 highly reproducible polymorphic bands. The ratio of discordant bands to total bands scored was used to estimate the genetic distance within and among populations. Multidimensional scaling (MDS) of the relation matrix showed distinctly separate clusters between New York and non-New York populations, indicating separation between these two groupings. The MDS analysis was confirmed using pooled chi-square tests for fragment homogeneity. This study shows that RAPD markers can be used as population-specific markers for Panax quinquefolium, and may eventually be utilized as markers for ginsenoside assessment.     

中药材蒲公英及其混淆品的DNA指纹鉴别研究

采用分子生物技术包括任意引物聚合酶链式反应（AP－PCR）和随意扩增多态性DNA（RAPD）方法扩增蒲公英及其六种土公英混淆品的基因组DNA，获得清晰可靠的DNA指纹图谱。根据琼脂糖胶上显示的DNA带型差异可鉴别蒲公英和土公英混淆品。     

鱼腥草种质资源的RAPD分析

目的分析鱼腥草种质资源在分子水平上的遗传多样性。方法应用RAPD技术对92份鱼腥草种质资源进行检测。结果34个随机引物中有32个引物(94.1%)扩增产物具多态性。34个引物共得到200条扩增DNA片段,其中93.5%的片段具多态性。每个多态性引物平均可扩增出5.8个多态性片段。峨眉蕺菜和蕺菜种内平均遗传相似系数(genetic similarity,GS)分别为0.521和0.572,二者种间GS值为0.517。峨眉蕺菜与蕺菜中染色体数目为36的细胞型间相似程度最高,其平均GS值达0.530。栽培蕺菜类群比其野生类群遗传多样性相对较高。聚类分析表明,利用RAPD技术可将全部供试材料区分开,所有材料共划分为14类。其中,绝大多数(62个)聚为一类,且根据RAPD遗传相似系数划分的类群同地理分布有一定关系。结论(1) 鱼腥草种质资源在分子水平上确实存在较大遗传差异。(2) RAPD标记可作为构建鱼腥草DNA指纹图谱的有效工具。(3) 鱼腥草药材道地性与环境因素有关,但更大程度上由其遗传因素所决定。     

利用随机扩增多态性DNA技术鉴定五种药用八角莲

现有的传统中药材鉴定方法有时很难可靠地用于药用植物八角莲的鉴定研究。本文在利用作者新建立的从干燥材料中分离高质量DNA的技术和比较不同聚合酶链反应（PCR）模板浓度的基础上,利用随机扩增多态性DNA（RAPD）技术对五种药用八角链的7个样品进行了分子鉴定。在筛选的42个引和中,12个引物产生的多态性条带,可靠地区分了这7个八角莲样品,包括3个不同地区的D.versipellis,1个D.majorense,1个D.pleiantha,1个D.furfuracea和1个D.veitchii。每个样品都找到了1－3个稳定可靠的特异分子标记。此外,本文发现D.furfuracea与D.versipellis的亲缘关系最远,与D.veitchii较近,而与D.makjorense最近。D.veitchii与D.majorense的亲缘关系比与D.versipellis,D.furfuracea和D.pleiantha更近。结果表明RAPD技术能够有效地用于这7个药用八角莲样品的分子鉴定。     

Genetic characterization and authentication of Embelia ribes using RAPD-PCR and SCAR marker

Embelia ribes Burm. f. (Myrsinaceae) is one of the important plants used in Indian traditional medicine. RAPD-PCR analysis was performed to obtain species-specific DNA fragments. A band of 906 bp that was specific to Embelia ribes irrespective of the geographical source was obtained using the random decamer primer OPF 05. SCAR primers ER 1 (27 mer) and ER 2 (26 mer) were designed from the sequence of the RAPD marker which yielded an expected amplicon of 594 bp with the Embelia ribes DNA only. This methodology can be used for species identification of genuine Embelia ribes and to distinguish it from common substitutes and adulterants. BLAST: basic local alignment search tool ER 1: Embelia ribes forward primer ER 2: Embelia ribes reverse primer RAPD-PCR: random amplification of polymorphic DNA polymerase chain reaction SCAR: sequence characterized amplified region.     

不同居群白芍的DNA随机扩增多态性研究

目的分析6个不同居群白芍的遗传多样性,为白芍的种质鉴定及遗传多样性分析提供依据。方法运用随机扩增多态性DNA(RAPD)技术,对浙江磐安、四川中江、安徽亳州、上海崇明、江苏宿迁和山东荷泽居群白芍的基因组DNA进行随机扩增,利用NTsys2.10e软件计算遗传相似性,运用UPGMA法进行聚类分析并构建树状图。结果共筛选了70个随机引物,从中挑选出8条多态性强、重复性好的引物,共检测出215个位点,多态性位点137个,多态位点比率为63.7%,UPGMA聚类可以将不同来源的白芍很好地区分开。结论不同产地间的白芍存在丰富的遗传多样性,RAPD分子标记方法可以用来鉴定不同产地的白芍。     

Identification of Anoectochilus formosanus and Anoectochilus koshunensis species with RAPD markers

RAPD (random amplified polymorphic DNA) markers were developed to distinguish Anoectochilus formosanus from Anoectochilus koshunensis and their putative hybrids. Morphological differentiation of these two species beyond the flowering period is difficult. RAPD markers provide a rapid and easy tool for identification of the two Anoectochilus species. In the study, forty arbitrary decamer primers were screened, and nineteen species-specific RAPD markers generated from polymerase chain reactions (PCR) with eight random primers were obtained. Nine were specific to A. formosanus and ten to A. koshunensis. Two primers, OPC-08 and OPL-07, produced two markers, one specific to A. formosanus and the other specific to A. koshunensis, which simultaneously appeared in the hybrids pattern. The RAPD markers can be applied both to identification of A. formosanus and A. koshunensis species and to assessment of the extent fo hybridization in hybrids between them. This information facilitates the breeding program process.     

中药材苦地胆及其混淆品的DNA指纹鉴定

用聚合酶链式反应方法，包括臆断引物聚合酶链式反应技术和随意引物扩增的多态性ＤＮＡ技术，鉴定中药材苦地胆及其混淆品，结果表明：用六个长的和一个短的引物扩增的苦地胆及其混淆品基因组ＤＮＡ指纹和多态性，可在琼脂糖凝胶上明显地区别开来。根据这些指纹图谱及由此估算的相似度值，证实从福建，台湾，香港以及澳门获得的商品药材苦地胆的基原为菊科植物地胆草。     

不同产地红曲基原菌的RAPD分析

目的：建立中药红曲基原菌的RAPD分析方法,方法：CTAB法提取不同产地红曲基真菌的基因组DNA,琼脂糖凝胶电泳法检测PCR扩增产物。PHYLIP3．5c进行聚类分析,结果：S282等10个随机引物的扩增产物谱带多,特征好,产地间指纹图谱呈现出明显的DNA扩增产物多态性,结论：RAPD分析技术可作为红曲鉴定的有用手段。